The genus Astragalus, family Leguminosae, comprises 2000 or more species widely distributed throughout the world, of which only certain plants, in the section Tragacantha, produce a polysaccharide rich exudate. Most of the polysaccharide gum producing species are indigenous to the Middle East countries from Eastern Turkey, Azerbaijan, Syria, Iraq and Iran to Western China. The gum from these Astragalus species is a commercial commodity called tragacanth. It is used in foods as a stabilizer, thickener and emulsifier, and in pharamaceutical products as a suspending agent. It is generally recognized as safe (GRAS) under Food and Drug Administration classification for use in foods in the United States. Other countries also classify tragacanth as safe for food additive use.
The gum is stored in the central cylinder of the tap root of the plant. It is collected from the live plant by cutting a slot or drilling a hole in the root cylinder below the crown, whereupon the gum exudes out as a ribbon. The gum dries within 1-2 hours becoming semi-brittle, and can be picked off by hand. The gum contains both water-soluble and insoluble polysaccharides and other minor components. The chemically heterogeneous whole dried exudates are marketed as tragacanth ribbon or powder. In the past it was estimated that as much as one million pounds of tragacanth per year were harvested from wild plants for food additive and pharmaceutical uses.
Roe (1959) found that some tragacanth powders inhibited the multiplication of cancer cells in the peritoneal cavity of mice. Suspended Landschutz ascites cancer cells were implanted in the peritoneal cavity of mice where they multiplied and could be assayed by counting techniques. The serous fluid of the peritoneal cavity was an ideal medium for the reproduction and growth of these transplanted ascites cancer cells. The ascites cancer cells were cultured in the peritoneal cavity and multiplied under normal conditions. When commercial tragacanth in water was injected into the peritoneal cavity the multiplication of cancer cells was inhibited. Roe assumed that the tragacanth had a direct cytostatic action on the cancer cells. The inhibition was explained by an association of the polysaccharide with the ascites cancer cell wall, with resultant inhibition of mitosis. This phenomena was examined in a series of Roe et al. articles following the initial report (Galbraith et al., 1962, 1963; Mayhew and Roe, 1964a, 1964b, 1965; Carr and Roe, 1967, Roe et al., 1972). In addition to the Landschutz strain, Roe worked with Crocker, Bp8 and C+ leukemia ascites cancer cells in the peritoneal cavity. She investigated tragacanth powders from what she believed was Astragalus gummifer a commonly used but inaccurate designation for Astragalus species producing the gum, and a type "tragacanth" from Indian Sterculia urens, which was actually karaya gum. These were the only two plant species mentioned by Roe in her studies of tragacanth.
Roe et al. (1972) reported their final study on the interaction of tragacanth with ascites cancer cells both in vivo and in vitro. In this study with commercial tragacanth, the mitosis inhibitory component was found to be located exclusively in the water-soluble fraction. As in the earlier work from this laboratory, the degree of inhibition of water-soluble fractions was assayed by counting the ascites cancer cells taken from the peritoneal cavity of an infected mouse. The inhibitory activity of the water-soluble fraction was destroyed by boiling for five minutes. In none of the Roe et al. reports were the studies ever extended to cancers that occur outside of the peritoneal cavity. Direct surface contact of ascites cancer cells with intraperitoneal administered tragacanth preparations was always postulated as the governing mechanism inhibiting mitosis. Effects on cell membrane rather than immunomodulatory activity were reported to be the cause of inhibition. These surface effects of the polysaccharide affecting the cancer cell directly in the peritoneal cavity may well have been the primary cause of mitosis inhibition in the Roe group studies. The Roe studies did not consider possible immunomodulatory activity that would lead to cancer inhibition of solid tumors outside of the peritoneal cavity.
Nakahara et al. (1964) studied the effects of several plant polysaccharides on the inhibition of a cancer implanted in the groin of mice. In this investigation Ehrlich ascites cancer cells were extracted from the peritoneal cavity of one mouse and injected subcutaneously into the groin of other mice where they grew into a solid tumor. After the tumor-bearing mice were administered polysaccharides intraperitoneally, growth of the solid tumor in the groin was monitored by sacrificing the mouse with observation and weighing of the tumor mass. Bamboo polysaccharide had an inhibitory effect on the growth of the tumor in the groin, but tragacanth did not. This study demonstrated that tragacanth when administered intraperitoneally did not act on a solid tumor outside of the peritoneal cavity.
Osswald (1968) repeated the experiments of Roe using Ehrlich ascites cells propagated in the peritoneal cavity of mice. Commercial tragacanth suspended in an aqueous glucose medium was injected into the peritoneal cavity of mice 6 and 24 hours prior to intraperitoneal injection of the ascites cancer cells. After seventeen days only 5 of 15 mice on the 6 hour pretreatment regimen developed new cancer cells, whereas 12 of 15 mice on the 24 hour pretreatment regimen developed new ascites cancer cells. Three of the thirty mice in the tragacanth treatment groups actually developed solid tumors in the stomach walls in addition to the ascites cancer cells in the peritoneal cavity. Administration of polysaccharides at high doses prior to intraperitoneal injection of ascites cancer cells as in this study is not an effective way to inhibit multiplication of the cells, and appears to partially inhibit the immune system facilitating metastasis.
In summary, Roe et al (1959-1972) and Osswald (1968), were only able to inhibit multiplication of ascites cancer cells transplanted into the peritoneal cavity of mice with their tragacanth preparations injected into the same site. Osswald confirmed Roe's finding about ascites cancer cell mitosis inhibition but found that tragacanth seemed to increase the spread of the cancer to the stomach wall tissue in some of the mice. Nakahara et al. (1964) was not able to inhibit the growth of a solid groin tumor in mice with intraperitoneal administration of tragacanth. These prior studies do not predict or anticipate the antiviral and anticancer results reported herein, which are based on an immunomodulatory mechanism of action rather than a direct effect of the polysaccharide on the antigenic cancer cell or virus.
Until this invention no autochthonous solid tumors have been inhibited by tragacanth preparations. Only transplanted serous fluid ascites cancer cells growing in the peritoneal cavity have been inhibited from multiplying. In further contrast to prior art, the purified polysaccharides of tragacanth reported here, although injected intraperitoneally, act on solid tumors outside of the peritoneal cavity. In the light of prior results there was no real basis to expect any tumors outside of the peritoneal cavity to be inhibited following intraperitoneal administration of tragacanth polysaccharide fractions, much less both chemical-induced and virus-induced tumors. There were also no examples in prior art which demonstrated immunomodulatory antiviral effects for these tragacanth polysaccharides.
Furthermore, the chemical-induced and virus-induced tumors of the mammary gland and spleen reported in this disclosure are not transplanted tumors that had a separate existance outside of the host animal. There were no cancer cells initiating the tumors in these test systems as there were in the prior ascites cancer cell reports. The chemical-induced and virus-induced solid tumors are considerably different from the transplanted serous fluid ascites tumors in this regard. The chemical-induced and virus-induced solid tumors are also different from one another, one establishing selectively in the mammary gland, and the other in the spleen of rodents. An extension of immunomodulatory activity against viruses further demonstrates in this disclosure that the polysaccharides modulate the immune system, and respond to various types of antigen challenges in a non-specific manner. In this regard, Seljelid et al. (1981) reported that gum tragacanth stimulates mouse macrophage in vitro. In the latter report it was claimed that the water-insoluble polysaccharide fractions stimulated macrophage, although not all insoluble polysaccharides. This is in contrast to our conclusion that water-soluble polysaccharides stimulate macrophage in the peritoneum, a rich source of these mononuclear phagocytes. Macrophage stimulation in the peritoneal cavity is an important mechanism leading to anticancer and antivirus effects away from this site of administration of these immunomodulatory plant polysaccharides.
The prior studies of Roe (1959-1972), Nakahara (1964) and Osswald (1968) were carried out principally with aqueous commercial tragacanth suspensions which included water-soluble, insoluble and gel fractions. Roe found that inhibitory activity varied in tragacanth from different areas but did not relate this to interspecies or intraspecies differences. The differences could have been due to processing or cultural conditions. In contrast, the differences in activity of five distinct Astragalus species are disclosed here. This is the first time that any antiviral and cancer inhibitory activity has been related to different Astragalus species. Each one of the Astragalus species, Tragacantha section, plants are different in appearance and growth habit from one another. Studies demonstrate that the tragacanth polysaccharides from each species are also different. The polysaccharides from three Astragalus species of Turkish origin (Anderson and Bridgeman, 1985) and four of Iranian origin, but U.S. grown (Anderson and Grant, 1989) were shown to differ in their contents of monosaccharides including galacturonic acid, galactose, arabinose, xylose, fucose and rhamnose. The polysaccharides also differ between species in their percent nitrogen, methoxyl and ash (metal) values, as well as amino acid composition of the minor amount of protein present. Differences in the amounts of water soluble and insoluble polysaccharides were also noted. Our studies found that acidity and viscosity of water-soluble tragacanth solutions, infrared spectra and size exclusion chromatography curves were also different among the species. Because of these differences in the gum exudates from various Astragalus species, it is not surprising to find differences in antiviral and tumor inhibitory activity among species. Each plant species varies in appearance, polysaccharide chemistry, and degree of biological activity from every other species within this Astragalus genus. All of the whole gums are named tragacanth and are thereby classified in the Tragacantha section but there are physical, compositional and immunomodulatory differences in polysaccharides among the species.
Anticarcinogenesis agents that prevent formation of tumors, lower multiplicity of tumors or size in original tissues, and prevent or inhibit the spread of the tumor to other tissues, can be useful medical tools. Such agents can be used as adjuncts to surgical, drug and radiation therapies, inhibiting metastasis. Many viral diseases will be susceptible to these polysaccharide immunomodulatory agents. The tragacanth products can also be used in the treatment of patients with immunological deficient systems. Tragacanth gum has been used in foods such as ice cream, sauces and dressings for many years as a generally recognized as safe (GRAS) food ingredient (Informatics, 1972). Gum tragacanth is non-teratogenic, non-mutagenic and generally non-toxic (Anderson, 1989). However, Bachman et al. (1978) found that commercial gum tragacanth administered to rats inhibited oxidative phosphorylation in liver and heart mitochondria, and mixed function oxidases in liver. It was speculated that the small molecular impurities in gum tragacanth could have caused these negative effects. The discoveries reported here extend the prior art into new, novel and safe plant polysaccharides for the treatment of diseases and disorders susceptible to immunomodulatory agents.